RESEARCH SUMMARY Tuberculosis (TB) has afflicted humans for roughly 70,000 years. Despite the advent of effective TB treatment options over 50 years ago, they are lengthy and complicated, and are directly associated with high frequency of treatment failure. The blunted efficacy of the current TB treatment is largely attributed to the ability of Mycobacterium tuberculosis (Mtb) to form persisters, a small fraction of phenotypic variants that are tolerant to antibiotic effects. This has been confirmed by mathematical modeling, which showed that prolonged treatment is required to ensure persister eradication. Thus, Mtb persisters constitute a therapeutically critical facet of the TB pandemic. However, little is known regarding the underlying metabolic processes through which Mtb forms persisters in an effort to survive drug treatment. PknG is one of 11 serine-threonine protein kinases and it monitors and corrects a perturbed cytoplasmic redox state. Accumulating evidence suggests that deleterious reactive oxygen species (ROS) are produced by antibiotics and target bacterial metabolism. Thus, ROS-mediated metabolic damage is a common microbicidal effector. As a countermeasure, Mtb has evolved adaptive metabolic mechanisms to circumvent antibiotic- mediated ROS production. Our preliminary data proved this by showing that Mtb seeks to avoid ROS damage, by maintaining essential metabolic activities such as the methylcitrate cycle (MCC) and TCA cycle for survival not by using external nutrients but by catabolically remodeling abundant endogenous mycolic acid. We also observed that PknG-mediated remodeling in glutamine-glutamate metabolism plays a crucial role in mitigating metabolic damage induced by overactive MCC. Taken together, inactivation of the MCC and/or PknG-mediated regulatory function impairs persister formation. Thus, we hypothesize that mycolic acid serves as an internal carbon reservoir to compensate for nutrient shortage due to limited exogenous carbon support, and that mycolic acid consumption requires regulatory crosstalk between PknG and MCC. The goals of this application are: to validate the relative contribution of mycolic acid consumption (Aim 1) and PknG-mediated metabolic remodeling for redox homeostasis (Aim 2) during persister formation and subsequent drug-tolerance. The outcomes of Aims 1 and 2 will be assessed as therapeutic options that can be used to enhance the efficacy of the current standard TB drug regimen by preventing persister formation, interfering with the immune-evasion strategy, and eradicating the metabolically synchronized Mtb (Aim 3). Our work will offer a new drug regimen that targets crosstalk between regulatory and catalytic circuits of Mtb metabolism. This new option will offer simpler and shorter treatment options that will lead to increasing patient compliance and cure rates, while decreasing the emergence of drug-resistant mutations.